GnRH is released in pulses and acts on the pituitary to stimulate the synthesis and release of LH and FSH. This pulsatility is crucial to maintain normal fertility. We previously demonstrated frequency-dependent, subunit-specific effects of GnRH pulses on rat gonadotropin gene transcription in vivo and in vitro, and now have GnRH-responsive luciferase (LUC) reporter gene constructs for the rat alpha and LHbeta genes. Frequency-dependent effects on transcription could arise from differential activation of Ca+2 and protein C(PCK)/mitogen-activated protein kinase (MAPK) cascade and/or divergent GnRH-responsive gene regions and transcription factors. The role of different intracellular signals in GnRH-stimulated endogenous gonadotropin gene transcription will be assessed by nuclear run-off assays in gonadotropes after treatment with specific activators of inhibitors of Ca+2 influx, PKC, and MAPK, in the absence or presence of GnRH. Pituitary cells from transgenic mice expressing LHbetaLUC will be similarly treated and Lhbeta promotor activity measured. Finally, alphaLUC and LHbetaLUC constructs will be transfected into clonal gonadotrope cell lines, and GnRH-stimulated activity measured in the absence or presence of inhibitors of calcium influx, MAPK, and with co-transfected dominant negative or constitutively active forms of MAPK and calcium-calmodulin kinase (CAMK). MAPK and CAMK enzyme activity will be measured directly in treated cells. GnRH- sensitive elements of the alpha and LHbeta genes will be identified by transfection analysis using deletion/mutation constructs of the reporter genes. Activity of the isolated DNA elements will be demonstrated on heterologous promoters, and DNA-nuclear protein binding performed by gels shift and footprinting assays and southwestern blot analysis. The ability of steroids to directly influence the response to GnRH, and binding of nuclear proteins to GnRH-sensitive gene regions will be determined. These studies will help define the underling mechanisms of frequency-dependent regulation of gonadotropin synthesis, and begin to identify specific cellular targets which confer the GnRH response.